Field: BIOTECHNOLOGY
Development and Mining Microsatellites for Crop Genome Resources
Harshvardhan N. Zala1, Tejas C. Bosamia2
1
Dept. of Agricultural Biotechnology, Anand Agricultural University, Anand-388 110
2
Dept. of Plant Biotechnology & Biotechnology, Junagadh Agricultural University, Junagadh-362 001
Microsatellites or simple sequence repeats (SSR)
are short (1–6 bp) repeat motifs associated with a
frequency of length polymorphism, found in
both coding and non-coding (5’UTR, 3’UTR,
introns) DNA sequences of all higher organisms
(Gupta et al., 1996). Variations in SSR regions
originate mostly from errors during the
replication process, frequently DNA polymerase
slippage. These errors generate base pair
insertions or deletions, resulting, respectively in
larger or smaller regions. SSRs have high-
mutation rates (between 10-2 and 10-6 mutations
per locus per generation, and on average 5×10-4)
that generate the high levels of allelic diversity.
Microsatellites determine the genetic functions
including gene regulation, development and
evolution. SSR markers have gained
considerable importance over other markers due
to many desirable attributes including
hypervariability, multiallelic nature, codominant
inheritance, reproducibility, relative abundance,
extensive genome coverage, chromosome
specific location, amenability to automation,
high throughput genotyping and their ability to
associate with many phenotypes (Parida et al.,
2009).
Classification of Microsatellites
SSRs have been variously classified (Kalia et al.,
2011)
1. Depending upon the number of
nucleotides per repeat unit
Mono-, di-, tri-, tetra-, penta- and hexa-
nucleotide repeats which represent (A)11, (GT)12,
(ATT)9, (ATCG)8, (TAATC)6 and (TGTGCA)5
respectively.
2. Arrangement of nucleotides in the repeat
motifs
(i) Perfect repeats are tandem arrays of a single
repeat motif ((CA)n) and further classified into
class I (≥ 20 nts) and class II (≥ 10 but < 20 nts)
repeats. (ii) Imperfect repeats are perfect repeats
interrupted by non-repeat motifs at some
locations ((CCA)n TT (CGA)n). (iii) Compound
repeats are two basic repeat motifs present
together in various configurations ((CA)n
(GA)n).
3. Genic and Genomic
Genic molecular markers (GMM) are developed
from coding sequences like expressed sequence
tags (ESTs) or fully characterized genes having
known functions and further classified as (i)
Gene-targeted markers (GTMs) and (ii)
Functional markers (FMs). Genomic molecular
markers are derived directly from the genomic
DNA and therefore could belong to either the
transcribed or the non-transcribed part of the
genome without any information available on
their functions.
4. Based on their location in the genome
(i) Nuclear SSRs (nuSSR) (ii) Mitochondrial
SSRs (mtSSR) (iii) Chloroplast SSRs (cpSSR)
Methods for development of microsatellites
There are several methods for developing SSR
markers (Zane et al., 2002)
1. Genomic DNA library: Colony
hybridization (BAC and BAC-end
sequencing)
2. Microsatellite enrichment: Microsatellite-
enriched library construction; Primer
extension and Selective hybridization (Fast
Isolation by AFLP of Sequences Containing
repeats-colony hybridization)
3. RAPD amplicons based methods: RAMPO,
RAHM, RAMS
4. Fingerprinting based methods: MP-
PCR/ISSR-PCR, REMAP, IRAP, AMP-
PCR, RAMP
1
5. Whole genome shotgun sequencing (WGS)
and WGS by hybridization with enrichment
probes
6. cDNA library construction and sequencing
7. Transcriptome sequencing through next
generation sequencing platforms like 454
Roche, Illumina, SoLiD
8. Transferability/Cross-species amplification:
Ability to effectively transfer SSR markers
across taxa
A particular microsatellite locus can be
identified by its flanking sequences, as the
sequences of flanking region are generally
conserved across individuals of the same species
and sometimes of different species, the SSR
markers can be utilized for amplification in that
species, making transferability a cost-effective
method of SSR development. Both genic and
genomic SSR markers can be transferred across
species, however, genic SSR markers are
expected to have a high transfer rate due to
conservation of transcribed regions among
related species.
9. Mining microsatellites from gene sequences:
Retrieving microsatellites from sequence
databases like NCBI or EMBL using
microsatellite search tools (softwares/
scripts). The expressed sequence tags (ESTs)
or gene sequences can be downloaded and
scanned for identification of SSRs by
employing several search modules or
programmes like MIcroSAtellite (MISA),
SSRfinder, Sputnik, SSR Identification Tool
(SSRIT), SSRSEARCH, Tandem Repeat
Finder (TRF) etc. for recognition of SSR
patterns.
SSR markers are designed from flanking
sequences of identified SSR motif by employing
primer designing softwares like Primer3,
BatchPrimer3, IDT etc. The designed primers
are checked for desired characteristics like
absence of dimerization, hairpin formation
(usually >3 bp) and low specific binding at 3'
end to avoid mispriming.
Validation of designed SSR markers
The in silico designed primers are verified for
amplification in parents of mapping population
or on a panel of distinct genotypes. The SSR
markers showing polymorphism are further
checked for polymorphism in segregating or
mapping population. Genic SSRs exhibit
comparatively less polymorphism than genomic
SSRs. Alleles that differ in length are
distinguished by high-resolution gel
electrophoresis, thus allowing rapid genotyping
of many individuals at many loci by sequencing
DNA. The microsatellite analysis will make use
of the differences in the number of repeats
between the different alleles.
Repertoire of genomic and genic SSRs have
been identified, developed and employed in a
variety of studies ranging from genotyping,
DNA fingerprinting, association mapping,
distant hybridization in several plant species
from cereals to legumes, vegetables to fruits,
medicinal and aromatic to plantation crops
(Varshney et al., 2007).
Applications
SSR markers find a wide range of applications
which includes genome mapping, population
genetics, diversity analysis, trait mapping,
functional diversity, interspecific or intergeneric
transferability, comparative mapping, marker-
assisted selection, genome wide association
mapping, hybrid testing, QTLs analysis, linkage
mapping, construction of high density genetic
maps, transcript map (genic SSRs) and
phylogenetic relationship.
Conclusion
SSR markers act as repository of valuable
genetic crop resources which can be exploited
for different molecular breeding approaches.
Various methods of development are employed
for SSR discovery along with transferability
analysis being highly explored for crops having
SSRs from related species. Mining of SSRs from
sequence data serves as a relatively easy and
cost-saving strategy for any organisms with
enough DNA data. Such approach demonstrates
in real time the efficiency of public data bases
put to use. Transcriptome sequencing through
NGS is gaining grounds for developing SSR
markers mainly due to availability of large
2
amount of data and ease in SSR development.
These genic and genomic SSR markers are also
utilized for saturating gaps in genetic maps.

Future Thrust
There is a need to characterize mapping
populations with QTL specific SSR markers to
facilitate comparative mapping studies, fine
mapping and integration of genetic maps in
several crops. Genic molecular markers should
be exploited for crops species, possessing less
polymorphism. Need to investigate SSR
transferability for assessing genetic variability
which can be used in wide hybridization to
introgress alien genes.

References
Gupta, P. K., Balyan, H. S., Sharma, P. C. and
Ramesh, B. (1996) Microsatellites in
plants: a new class of molecular
markers. Curr Sci, 70: 45–54.
Kalia, R. K., Rai, M. K., Kalia, S., Singh, R. and
Dhawan, A. K. (2011) Microsatellite
markers: an overview of the recent
progress in plants. Euphytica, 177:309–
334.
Parida, S. K., Kalia, S. K., Sunita, K., Dalal, V.,
Pandit, A., Singh, A., Sharma, T. R. and
Singh, N. K. (2009) Informative
genomic microsatellite markers for
efficient genotyping applications in
sugarcane. Theor Appl Genet, 118:327–
338.
Varshney, R. K., Mahender, T., Aggrawal, R. K.,
Borner, A. (2007) Genic molecular
markers in plants: development and
applications. In: Genomics assisted crop
improvement, Vol I: Genomics
Approaches and Platforms (Eds
Varshney RK, Tuberosa R), Springer,
Dordrecht, The Netherlands, pp 13-30.
Zane, L., Bargelloni, L. and Patarnello, T. (2002)
Strategies for microsatellite isolation: A
review. Mol Ecol. 11:1–16.